阅读说明：本技术 一种基于碳球负载二硫化钼掺杂铜铑双金属构建的电化学免疫传感器的制备及应用 (Preparation and application of electrochemical immunosensor constructed based on carbon sphere loaded molybdenum disulfide doped copper rhodium bimetal ) 是由 鞠熀先 丁慧 魏琴 贾洪英 邵明月 范大伟 孙旭 曹伟 王雪莹 匡轩 于 2019-10-29 设计创作，主要内容包括：本发明涉及一种基于碳球负载二硫化钼掺杂铜铑双金属构建的免疫传感器的制备及应用,属于纳米材料和电化学分析技术领域。采用铜铑双金属改性的碳球负载二硫化钼制备电化学免疫传感器,用于降钙素原的灵敏检测。(The invention relates to preparation and application of an immunosensor constructed based on carbon sphere loaded molybdenum disulfide doped copper rhodium bimetal, and belongs to the technical field of nano materials and electrochemical analysis. The electrochemical immunosensor is prepared by adopting copper-rhodium bimetallic modified carbon spheres loaded with molybdenum disulfide and is used for sensitive detection of procalcitonin.)

1. A preparation method of an electrochemical immunosensor constructed on the basis of carbon sphere loaded molybdenum disulfide doped copper rhodium bimetal is characterized by comprising the following preparation steps:

(1) preparation of mesoporous carbon spheres

1) Adding 3 ~ 15 mL of tetraethyl orthosilicate into a mixed solution of 70 ~ 160 mL of ethanol, 10 ~ 50 mL of water and 3 ~ 15 mL of ammonia water, magnetically stirring for 15min, then sequentially adding 0.4 ~ 1.8.8 g of resorcinol and 0.5 ~ 3 mL of formaldehyde solution, and stirring for 24h at room temperature;

2) respectively washing the mixture for three times by using ultrapure water and absolute ethyl alcohol after stirring to remove impurities, and finally placing the collected product in a vacuum drying box for drying at 70 ℃ for 12 hours;

3) calcining the dried product in a nitrogen environment at 700 ℃ for 5 hours;

4) etching the calcined product by using a sodium hydroxide solution, washing the calcined product by using ultrapure water to remove impurities, placing the collected product in a vacuum drying oven, and drying the product for 12 hours at 70 ℃ to obtain a product mesoporous carbon sphere;

(2) preparation of carbon sphere loaded molybdenum disulfide

1) Sequentially adding 20 ~ 60 mg of mesoporous carbon spheres and 10 ~ 40 mg of ammonium tetrathiomolybdate into 30 mL of N, N-dimethylformamide solution, and carrying out ultrasonic treatment for 30 min;

2) continuously adding 1 ~ 5 mL of hydrazine hydrate into the mixed solution, and carrying out ultrasonic treatment for 30 min;

3) continuously transferring the solution into a 50 mL high-pressure reaction kettle, and reacting for 18 h at 210 ℃;

4) after the reaction is finished, cooling the solution to room temperature, washing the solution with ultrapure water and absolute ethyl alcohol for three times respectively, and collecting the obtained product for freeze drying;

(3) preparation of copper rhodium bimetallic

1) Adding 8 ~ 30 mg of rhodium acetylacetonate and 7 ~ 25 mg of copper acetylacetonate into a mixed solution of 2 ~ 10 mL of oleylamine and 5 ~ 20 mL of toluene in turn, and stirring for 10 min;

2) continuously adding 20 ~ 80 mg of dimethylamine borane into the solution, and stirring for 20 min;

3) transferring the solution into a 50 mL high-pressure reaction kettle, and reacting for 12 h at 190 ℃;

4) after the reaction is finished, cooling the solution to room temperature, washing the solution with cyclohexane and absolute ethyl alcohol for three times respectively, collecting the obtained product, and dispersing the product into 2 mL of water for later use;

(4) preparation of carbon sphere loaded molybdenum disulfide doped copper rhodium bimetallic compound

1) Mixing 2 ~ 8 mL of copper-rhodium bimetallic solution with 2 mg of aminated carbon sphere loaded molybdenum disulfide, oscillating for 12 h, and placing in a refrigerator at 4 ℃ for later use after oscillation is finished;

(5) preparation of electrochemical immunosensor

1) Polishing the glassy carbon electrode to a mirror surface by using 0.05 mm, 0.3 mm and 1.0 mm of aluminum oxide powder in sequence, and cleaning by using ultrapure water;

2) dripping 6.0 mu L of 0.5 ~ 2.5.5 mg/mL carbon sphere loaded molybdenum disulfide doped copper rhodium bimetallic compound solution onto the surface of an electrode, and storing in a refrigerator at 4 ℃ until the solution is dried;

3) continuously dropwise adding 6 mu L of procalcitonin antibody 6 ~ 12 mu g/mL to modify the procalcitonin antibody on the surface of the electrode, and storing in a refrigerator at the temperature of 4 ℃ until the procalcitonin antibody is dried;

4) continuously dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 0.5% and ~ 2% to seal nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution with the pH =7.38, and storing in a refrigerator at 4 ℃ until the electrode is dried;

5) respectively dropwise adding 6 mu L of procalcitonin antigen with different concentrations to modify the surface of the electrode, incubating for 1 h at the temperature of 4 ℃, washing the surface of the electrode by using a phosphate buffer solution with the pH =7.38, and storing in a refrigerator at the temperature of 4 ℃ until the surface is dried.

2. The preparation method of the electrochemical immunosensor, according to claim 1, characterized in that the carbon sphere loaded molybdenum disulfide and the copper rhodium bimetal are synthesized by a simple hydrothermal synthesis method, and the copper rhodium bimetal is firmly bonded to the surface of the aminated carbon sphere loaded molybdenum disulfide by means of oscillation.

3. The preparation method of claim 1 is used for preparing an electrochemical immunosensor based on carbon sphere loaded molybdenum disulfide doped copper rhodium bimetal for detecting procalcitonin.

4. The use of an electrochemical immunosensor according to claim 3 as a procalcitonin assay, comprising the following steps:

(1) the test in the experimental process adopts a three-electrode system, wherein a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the constructed sensor is a working electrode, and the whole experiment is carried out in 10 mL of 50 mmol/L phosphate buffer solution with the pH value of 6.24 ~ 8.04.04;

(2) detecting the analyte by a time-lapse current method, wherein the input voltage is-0.4V, and the running time is 400 s;

(3) when the background current tends to be stable, 10 mu L of hydrogen peroxide solution with the concentration of 5 mol/L is injected into 10 mL of phosphate buffer solution with the concentration of 50 mmol/L and the pH value of 6.24 ~ 8.04.04 every 90 s, and the change of the current is recorded;

(4) and replacing the procalcitonin sample solution to be detected with the procalcitonin standard solution for detection.

Technical Field

The invention relates to preparation and application of an electrochemical immunosensor constructed based on carbon sphere loaded molybdenum disulfide doped copper rhodium bimetal, and belongs to the field of nano materials and electrochemical analysis.

Background

Procalcitonin (PCT) is a protein secreted by thyroid C cells, and the concentration of PCT in humans reflects the activity of the systemic inflammatory response, a sensitive biomarker of inflammation. PCT levels are elevated when there is severe infection, fungal, parasitic infection, as well as sepsis and multi-organ failure in the organism. In clinical tests, antibiotic treatment is strongly recommended when the concentration of PCT in the serum exceeds 0.5 ng/mL. The PCT content in the serum also has a guiding effect on the diagnosis of the sepsis, and the PCT higher than 2ng/mL indicates that the patient is infected with the sepsis. Therefore, accurate detection of PCT in serum is crucial to early diagnosis of sepsis, and has important significance in further guidance of therapy, and how to realize efficient and accurate detection of procalcitonin is a problem to be solved at present.

Electrochemical analysis methods have attracted much attention because of their advantages such as high sensitivity, high specificity, easy operation, and easy miniaturization. In recent years, the basic principle of an electrochemical immunosensor, which combines an electrochemical analysis method and a biological immunosensor technology, is based on specific recognition among antigen and antibodies, and has the advantages of high response speed, low cost and the like. Therefore, the method has good development and application prospects.

The invention discloses a carbon sphere loaded molybdenum disulfide-based electrochemical immunosensor, which is constructed by adopting a carbon sphere loaded molybdenum disulfide-doped copper rhodium bimetal as a sensor platform and is used for detecting procalcitonin. Molybdenum disulfide is a typical layered structure, six sulfur atoms are coated around each molybdenum atom, the molybdenum atom presents a triangular prism shape, and the prism surface of the molybdenum disulfide can also be used as a catalytic active center. Studies have shown that molybdenum disulfide has catalytically active sites with high surface energy on the edge planes. Molybdenum disulfide is considered a potentially desirable alternative to platinum group catalysts due to its high degree of electrocatalytic activity and binding energy with atomic hydrogen. In addition, the molybdenum disulfide can be effectively combined with other nano materials, and shows excellent compatibility. In the experiment, the carbon sphere-loaded molybdenum disulfide is synthesized by a hydrothermal synthesis method, so that the contact specific surface area is increased, and the carbon sphere-loaded molybdenum disulfide can have more active sites. Secondly, copper-rhodium bimetal is doped in the experiment, so that the copper-rhodium bimetal catalyst has a synergistic effect and shows a better catalytic effect on hydrogen peroxide. The carbon sphere loaded molybdenum disulfide copper rhodium doped bimetallic composite material prepared by the invention has the following advantages: the carbon sphere loaded molybdenum disulfide has large specific surface area and excellent catalytic performance, the introduced copper rhodium bimetal can be uniformly distributed on the surface of the carbon sphere loaded molybdenum disulfide, the electron transfer rate is accelerated through the synergistic effect between the copper rhodium bimetal and the carbon sphere loaded molybdenum disulfide, the catalytic activity of the composite material is improved, finally, the synthesized material has excellent catalytic capability on hydrogen peroxide, and the copper rhodium bimetal can also greatly improve the loading capacity of an antibody.

Disclosure of Invention

One purpose of the invention is to provide a method for preparing carbon sphere loaded molybdenum disulfide based on a hydrothermal method;

the invention also aims to prepare the copper-rhodium bimetallic composite material doped with molybdenum disulfide loaded on the carbon spheres, and the aminated material provides more amino groups and constructs stable amido bonds with carboxyl groups on the antibody to increase the immobilization of the antibody;

the immunosensor prepared based on the carbon sphere loaded molybdenum disulfide doped copper rhodium bimetal is applied to detection of procalcitonin in serum, the aminated copper rhodium bimetal can be directly connected with a procalcitonin antibody, other connecting agents are not needed, the operation steps of an experiment are simplified, the detection limit of the procalcitonin is widened, the detection range is enlarged, and the detection sensitivity is greatly improved.

The technical scheme of the invention is as follows:

1. preparation method of electrochemical immunosensor based on carbon sphere loaded molybdenum disulfide doped copper rhodium bimetal

(1) Preparation of mesoporous carbon spheres

1) Adding 3 ~ 15 mL of tetraethyl orthosilicate into a mixed solution of 70 ~ 160 mL of ethanol, 10 ~ 50 mL of water and 3 ~ 15 mL of ammonia water, magnetically stirring for 15min, then sequentially adding 0.4 ~ 1.8.8 g of resorcinol and 0.5 ~ 3 mL of formaldehyde solution, and stirring for 24h at room temperature;

2) respectively washing the mixture for three times by using ultrapure water and absolute ethyl alcohol after stirring to remove impurities, and finally placing the collected product in a vacuum drying box for drying at 70 ℃ for 12 hours;

3) calcining the dried product for 5 hours in a nitrogen environment at 700 ℃;

4) etching the calcined product by using a sodium hydroxide solution, washing the calcined product by using ultrapure water to remove impurities, placing the collected product in a vacuum drying oven, and drying the product for 12 hours at 70 ℃ to obtain a product mesoporous carbon sphere;

(2) preparation of carbon sphere loaded molybdenum disulfide

1) Sequentially adding 20 ~ 60 mg of mesoporous carbon spheres and 10 ~ 40 mg of ammonium tetrathiomolybdate into 30 mL of N, N-dimethylformamide solution, and carrying out ultrasonic treatment for 30 min;

2) continuously adding 1 ~ 5 mL of hydrazine hydrate into the mixed solution, and carrying out ultrasonic treatment for 30 min;

3) continuously transferring the solution into a 50 mL high-pressure reaction kettle, and reacting for 18 h at 210 ℃;

4) after the reaction is finished, cooling the solution to room temperature, washing the solution with ultrapure water and absolute ethyl alcohol for three times respectively, and collecting the obtained product for freeze drying;

(3) preparation of copper rhodium bimetallic

1) Adding 8 ~ 30 mg of rhodium acetylacetonate and 7 ~ 25 mg of copper acetylacetonate into a mixed solution of 2 ~ 10 mL of oleylamine and 5 ~ 20 mL of toluene in turn, and stirring for 10 min;

2) continuously adding 20 ~ 80 mg of dimethylamine borane into the solution, and stirring for 20 min;

3) transferring the solution into a 50 mL high-pressure reaction kettle, and reacting for 12 h at 190 ℃;

4) after the reaction is finished, cooling the solution to room temperature, washing the solution with cyclohexane and absolute ethyl alcohol for three times respectively, collecting the obtained product, and dispersing the product into 2 mL of water for later use;

(4) preparation of carbon sphere loaded molybdenum disulfide doped copper rhodium bimetallic compound

1) Mixing 2 ~ 8 mL of copper-rhodium bimetallic solution with 2 mg of aminated carbon sphere loaded molybdenum disulfide, oscillating for 12 h, and placing in a refrigerator at 4 ℃ for later use after oscillation is finished;

(5) preparation of electrochemical immunosensor

1) Polishing the glassy carbon electrode to a mirror surface by using 0.05 mm, 0.3 mm and 1.0 mm of aluminum oxide powder in sequence, and cleaning by using ultrapure water;

2) dripping 6.0 mu L of 0.5 ~ 2.5.5 mg/mL carbon sphere loaded molybdenum disulfide doped copper rhodium bimetallic compound solution onto the surface of an electrode, and storing in a refrigerator at 4 ℃ until the solution is dried;

3) continuously dropwise adding 6 mu L of procalcitonin antibody 6 ~ 12 mu g/mL to modify the procalcitonin antibody on the surface of the electrode, and storing in a refrigerator at the temperature of 4 ℃ until the procalcitonin antibody is dried;

4) continuously dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 0.5% and ~ 2% to seal nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution with the pH =7.38, and storing in a refrigerator at 4 ℃ until the electrode is dried;

5) respectively dropwise adding 6 mu L of procalcitonin with different concentrations to modify the surfaces of the electrodes, incubating for 1 h at the temperature of 4 ℃, washing the surfaces of the electrodes by using a phosphate buffer solution with the pH =7.38, and storing in a refrigerator at the temperature of 4 ℃ until the surfaces are dried.

2. The application of the electrochemical immunosensor for detecting procalcitonin based on the carbon sphere loaded molybdenum disulfide doped copper rhodium bimetallic compound is provided.

3. The electrochemical immunosensor is used for procalcitonin detection, and the detection steps are as follows:

(1) the test in the experimental process adopts a three-electrode system, wherein a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the constructed sensor is a working electrode, and the whole experiment is carried out in 10 mL of 50 mmol/L phosphate buffer solution with the pH value of 6.24 ~ 8.04.04;

(2) detecting the analyte by a time-lapse current method, wherein the input voltage is-0.4V, and the running time is 400 s;

(3) when the background current tends to be stable, 10 mu L of hydrogen peroxide solution with the concentration of 5 mol/L is injected into 10 mL of phosphate buffer solution with the concentration of 50 mmol/L and the pH value of 6.24 ~ 8.04.04 every 90 s, and the change of the current is recorded;

(4) the method comprises the steps of replacing a procalcitonin standard solution with a procalcitonin sample solution to be detected for detection, and detecting procalcitonin based on an electrochemical immunosensor of molybdenum disulfide-doped copper rhodium loaded on a carbon ball, wherein the detection range is 0.001-100 ng/mL, and the detection limit is 1.09 pg/mL.

Advantageous results of the invention

(1) The carbon sphere loaded molybdenum disulfide is used as a substrate, so that the carbon sphere loaded molybdenum disulfide has a large specific surface area and good electron transfer capacity, and has excellent catalytic performance on hydrogen peroxide;

(2) the doped copper-rhodium bimetal has good conductivity and biocompatibility, and the synergistic effect between metals is beneficial to fixing a large amount of antibodies, so that the sensitivity of the sensor can be greatly improved;

(3) the invention discloses a carbon sphere loaded molybdenum disulfide doped copper rhodium bimetallic electrochemical immunosensor which is prepared and used for detecting procalcitonin. The method has the advantages of simple reaction process, high response speed and wide signal response range, shows linearity in the range of 0.001-100 ng/mL, has the detection limit of 1.09 pg/mL, and realizes simple, quick and high-sensitivity detection of procalcitonin.

Detailed description of the preferred embodiments